Helicobacter pylori sialic acid binding adhesin, saba and saba-gene

ABSTRACT

An isolated  Helicobacter pylori  protein binding to sialyl-Lewis x antigen and having an approximate molecular weight of 66 kDa and comprising the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and sialyl-Lewis x antigen-binding  H. pylori  alleles of the protein, recombinant forms of the protein, such as a protein having the amino acid sequence SEQ ID NO: 5, or the protein alleles, and sialyl-Lewis x antigen binding portions of the proteins, are disclosed. The protein or portion of protein maybe used as a medicament or diagnostic antigen, and can be used in a method of determining the presence of sialyl-Lewis x antigen-binding  H. pylori  bacteria in a biological sample. Further, a DNA molecule encoding the protein or portion of protein, a vector comprising the DNA molecule, and a host transformed with the vector are comprised by the disclosure. Additionally, a method of determining the presence of sialyl-Lewis x or related carbohydrate structures in a sample, is described. This method has a wide range of different applications.

[0001] The present invention relates to a Helicobacter pylori Sialicacid binding Adhesin, SabA and sabA—gene. In particular, the inventionrelates to an isolated Helicobacter pylori protein binding tosialyl-Lewis x antigen and having an approximate molecular weight of 66kDa. The protein, or a sialyl-Lewis x antigen binding portion of theprotein, may be used as a medicament or diagnostic antigen, and it canbe used in a method of determining the presence of sialyl-Lewis xantigen-binding H. pylori bacteria in a biological sample. The inventioncomprises also a DNA molecule encoding the protein or a sialyl-Lewis xantigen binding portion of the protein, a vector comprising the DNAmolecule, and a host transformed with the vector.

BACKGROUND

[0002]Helicobacter pylori is considered the causative agent of chronicactive gastritis and peptic ulcer disease (Marshall and Warren, 1984),and is also correlated to development of gastric cancer (Parsonnet,1998). H. pylori colonizes the human gastric epithelial lining and themucus layer of primates and humans. For adherence, bacteria expressattachment molecules (adhesins) that bind specifically to cell surfaceproteins and glycoconjugates i.e., the receptors (Hultgren et al.,1993). Thus, the adhesins will target the infection to a limited numberof hosts, tissues and cell lineages (Karlsson, 1998).

[0003] We have previously demonstrated H. pylori adherence to thefucosylated blood group antigen H1 and Lewis b (Borén et al., 1993). TheH-antigen is typically found on erythrocytes defining the O phenotype inthe ABO blood group system, but the fucosylated histo-blood groupantigens are also expressed on the epithelial cell surfaces along thegastrointestinal tract (Clausen et al., 1989). Individuals of bloodgroup O phenotype are common among patients suffering from peptic ulcerdisease (discussed in Borén et al., 1994). Recently we found that theLewis b antigen binding property is prevalent among the virulent strainsthat carry the cag-Pathogenicity Island and the vacuolating cytotoxini.e., triple-positive strains. We therefore propose that Lewis b antigenmediated adherence of H. pylori plays a critical role for development ofsevere disease (Gerhard et al., 1999). Adherence of H. pylori to thegastric epithelial lining was recently demonstrated in the transgenicLewis b mouse expressing human α1,¾ fucosyltransferase (Falk et al.,1995). Challenge experiments suggest that H. pylori adherence mediatedby the Lewis b antigen activate inflammatory responses (Guruge et al.,1998).

[0004] In order to identify the Lewis b antigen binding H. pyloriadhesin we developed the Retagging-technique (Ilver-Arnqvist et al.,1998). The blood group antigen binding adhesin, BabA, belongs to afamily of outer membrane proteins (Tomb et al., 1997). We havepreviously shown that a babA-mutant strain although totally devoid ofLewis b antigen binding propeties, still adheres avidly to the humangastric epithelial lining (WO 00/56343). We have also previouslyidentified the sialyl-dimeric-Lewis x glycosphingolipid to which thebabA-mutant strain adheres with high affinity (WO 00/56343).

DESCRIPTION OF THE INVENTION

[0005] The present invention provides a sialic acid binding adhesin,SabA, binding the sialyl-Lewis x antigen. SabA was identified andpurified from the Helicobacter pylori babA-mutant by theRetagging-technique and it binds to the sialyl dimeric-Lewis xglycosphingolipid to which the babA-mutant strain adheres (WO 00/56343).Our new results suggest a flexible adaptation of bacterial adherenceproperties by alternative adherence modes and adhesins, to meet variousinflammatory responses, such as defensive shifts in the detailedglycosylation patterns of the gastric mucosa and the epithelial lining,during the course of chronic infectious disease.

[0006] The present invention is particularly directed to an isolatedHelicobacter pylori protein binding to sialyl-Lewis x antigen and havingan approximate molecular weight of 66 kDa (i.e. the actual molecularweight may be up to 10 % higher) and comprising the amino acid sequencesSEQ ID NO:1, QSIQNANNIELVNSSLNYLK, SEQ ID NO:2, IPTINTNYYSFLGTK, SEQ IDNO:3, YYGFFDYNHGYIK, and SEQ ID NO:4, DIYAFAQNQK,

[0007] and sialyl-Lewis x antigen-binding H. pylori alleles of theprotein and recombinant forms of the protein, such as SEQ ID NO: 5, orthe protein alleles, or sialyl-Lewis x antigen binding portion of theproteins. The recombinant proteins are thus expressed from a geneencoding the sialyl-Lewis x antigen-binding protein or the alleles.

[0008] The alleles of the protein may have an amino acid sequence thatdiffers from the isolated H.pylori protein with up to 15%, normallyabout 10% or less, such as 5%, but they shall have sialyl-Lewis xantigen-binding properties to be comprised by the present invention.

[0009] The recombinant forms of the protein may have the amino acidsequence of the full length isolated protein or its alleles or may havean amino acid sequence that corresponds to a sialyl-Lewis x antigenbinding fragment of the isolated protein or one of its alleles or anoptimized amino acid sequence with regard to production requirementsand/or immunizing properties.

[0010] The invention is also directed to the use of a protein or asialyl-Lewis x antigen binding portion of a protein comprised by theinvention for use as a medicament. The medicament may be used forinhibition of H. pylori binding to human tissues since the proteins orsialyl-Lewis x antigen parts of the proteins of the invention bind tohuman or animal glycoconjugates presented on patient's tissues. Further,the medicament may be a therapeutic or prophylactic vaccine againstHelicobacter pylori infection, wherein the protein is an activeingredient, optionally together with other active ingredients, such asother Helicobacter pylori antigenic proteins. The formulations of themedicaments or vaccines of the invention will be decided by themanufacturer using Good Manufacturing Procedure accepted by the medicalauthorities. The doses administered to patients will be decided by thepatient's physician based on recommendations from the manufacturer.

[0011] The invention is further directed to a diagnostic antigen for theimmunological determination, in a biological sample, of antibodiesagainst sialyl-Lewis x antigen-binding protein, wherein the diagnosticantigen is an optionally labeled protein or a sialyl-Lewis x antigenbinding portion of a protein comprised by the present invention.Examples of the biological sample are a biopsy, blood or plasma sample,and examples of immunological determinations are ELISA-assays andRIA-assays. Thus, the proteins and the sialyl-Lewis x antigen-bindingportions of the proteins of the invention may be conjugated to areporter molecule, such as a fluorescent marker, radiolabelling or anenzyme producing a detectable signal or biotin or other affinity tag toenable recognition of the labeled molecule of the invention.

[0012] Another aspect of the invention is directed to a method ofdetermining the presence of sialyl-Lewis x antigen-binding H. pyloribacteria in a biological sample, which comprises an immunologicaldetermination of the presence of antibodies binding to an optionallylabeled protein comprised by the invention. An example of the biologicalsample is a biopsy sample.

[0013] The invention is also directed to a DNA molecule encoding aprotein or a sialyl-Lewis x antigen binding portion of a proteinaccording to the invention, a vector comprising the DNA molecule, and ahost transformed with the vector. The DNA molecule may be isolated orsynthetic and will only code for a protein or part of the protein of theinvention. The vector may comprise, in addition to the DNA molecule ofthe invention, genes or gene fragments for the construction of fusionproteins, e.g. recombinant SabA-fusion proteins for different purposes.The vector of the invention is preferably a plasmid, and the host ispreferably a microorganism. The DNA molecule, the vector and the hostare useful in the production of a recombinant protein or a sialyl-Lewisx antigen binding portion of a protein comprised by the invention.Methods of producing recombinant proteins are well-known to a manskilled in the art of biotechnology.

[0014] Yet another aspect of the invention is directed to a method ofdetermining the presence of sialyl-Lewis x or related carbohydratestructures in a sample, comprising bringing the sample into contact withan optionally labelled protein or sialyl-Lewis x antigen binding portionof a protein according to claim 1 or 2, allowing binding of the proteinor sialyl-Lewis x antigen binding portion of the protein according toclaim 1 or 2 to the carbohydrate structure and determining the presenceof sialyl-Lewis x or related carbohydrate structures in the sample bydetermining

[0015] a) the occurrence of the binding, or

[0016] b) the absence of binding in case an analyte inhibiting thebinding is present.

[0017] The binding of the proteins and the sialyl-Lewis xantigen-binding portions of the proteins of the invention or theirlabeled molecules to carbohydrate structures, in particular sialyl-Lewisx and related carbohydrates, can be used for several applications, suchas diagnostic purposes, for protein purification, screening ofsubstances which bind to proteins and the sialyl-Lewis x antigen-bindingportions of the proteins of the invention including human and animalglycoconjugates, and to detect receptors for H. pylori or pathologicchanges of the tissue. Preferably the tissue or sample or preparation oftissue is from human gastric tissue or from human tumor tissue.Therefore, the proteins and the sialyl-Lewis x antigen-binding portionsof the proteins of the invention can be used in a method of diagnosing adisease, preferably a gastric disease, cancer or an inflammatorydisease.

[0018] The proteins and the sialyl-Lewis x antigen-binding portions ofthe proteins of the invention can be used in assays to determine, e.g.by measurement, the binding to the proteins and the sialyl-Lewis xantigen-binding portions of the proteins of the invention ofcarbohydrates, such as sialyl-Lewis x and other carbohydrate substancesor carbohydrate analog substances. Such assays may measure a) directbinding of the proteins and the sialyl-Lewis x antigen-binding portionsof the proteins of the invention to carbohydrates or b) inhibition bythe analyte of binding of a proteins and the sialyl-Lewis xantigen-binding portions of the proteins of the invention to acarbohydrate ligand. The assays may be performed in solution by use ofe.g. NMR or in solid phase in numerous formats in which the proteins andthe sialyl-Lewis x antigen-birding portions of the proteins of theinvention or their ligands can be immobilized. The assays to determinebinding to the proteins and the sialyl-Lewis x antigen-binding portionsof the proteins of the invention to carbohydrates such as sialyl-Lewis xand other carbohydrate substances or carbohydrate analog substances canbe used to screen combinatorial libraries of carbohydrate molecules andanalogs thereof. Methods to produce combinatorial libraries andcombinatorial carbohydrate or glycoconjugate libraries are well-known inthe art.

[0019] The invention will now be illustrated by description ofexperiments and drawings, but the scope of protection is not intended tobe limited to the specific disclosures.

DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1. is a diagram which shows that H. pylori strains bind thesialyl-Lewis x antigen with high affinity.

[0021] (A) H. pylori strains were analyzed for binding to ¹²⁵I-labeledneoglycoconjugates. Bacterial binding is given by the bars, from left tothe right; The Lewis b-, sialyl-Lewis x-, sialyl-Lewis a-, and sialyl-α2,3 lactose- all albumin conjugates.

[0022] (B) The 101 Swedish H. pylori strains were analyzed forneuraminidase dependent hemagglutination (HA), here shown with medianvalues indicated in the boxplots. A strong correlation according toPearson was found between the shift(s) in HA titers after sialidasetreatment of the red blood cells (removal of sialic acid residues) andbacterial binding of the soluble ¹²⁵I-labeled sLex antigen; 0.58,p=0.000.

[0023]FIG. 2. shows the Retagging of the sialic acid binding adhesin,SabA, and identification of the corresponding gene, JHP622.

[0024] (A) The sialyl-Lewis x antigen was used with the Retaggingtechnique for identification of the corresponding adhesin, in thebabA1babA2-mutant. After contact dependent Retagging and biotintransfer, the 66 kDa biotin tagged adesin SabA, was identified bySDS-PAGE, and subjected to MALDI-TOF. As a control, the Lewis b antigenwas used to Retagg the 17875 (wild type) strain, which thus visualizedthe 75 kDa BabA adhesin.

[0025] (B) All four peptide sequences were identified by Q-TOF andaligned with the deduced amino acid sequence of the chromosomal JHP662gene (SEQ ID NO: 5)(4 peptide matches (two unique grey bars and the twocommon black bars)) and the deduced amino acid sequence of thechromosomal JHP659 gene (SEQ ID NO: 6) (2 peptide matches (the twocommon black bars)).

DESCRIPTION OF EXPERIMENTS

[0026] Experimental Procedures

[0027] The procedures described herein are based on previously publishedteachings, and therefore the teachings of the herein cited publicationsare incorporated herein by reference.

[0028] Strains and Growth Conditions

[0029]H. pylori strains 26695 (Tomb et al., 1997), J99 (Alm et al.,1999), CCUG17875, and the babA-mutant stain were recently described(Ilver-Arnqvist et al., 1998). The 17875babA1::kan babA2::cam(double)-mutant strain was described in WO 00/56343. H. pylori clinicalisolates were from the University Hospital in Uppsala, Sweden. Bacteriawere grown at 37° C. in 10% CO₂ and 5% O₂, for 2 days.

[0030]H. pylori Binding to Neo-Glycoconjugates

[0031]¹²⁵I-labeled sialyl-α2,3lactose-, sialyl-Lewis a-, sialyl-Lewis x-and Lewis b-neoglyco-conjugates (IsoSep AB, Tullinge, Sweden) bound tobacteria were measured by gamma counting. Binding experiments werereproducible and performed in triplicates. RIA and Scatchard analyseswere performed essentially as described in Ilver-Arnqvist et al., 1998.

[0032] Sialidase-Dependent Hemagglutination of H. pylori

[0033] Erythrocytes (RBC) were obtained by vein puncture from a healthydonor and were washed with PBS and used at 0.75% (v/v) concentration.Sialidase treatment of RBC was performed as described (Paulson et al,1987) using Vibrio cholerae sialidase. Preparation of bacterial samples,titration and haemagglutination assays were performed as describedbefore (Hirmo et al., 1996) on microtiter plates.

[0034] Purification and Identification of the SabA Adhesin by Retagging.

[0035] The SabA adhesin was purified as previously described for theBabA adhesin (Ilver-Arnqvist, et al., 1998), with some modifications. H.pylori was incubated with sialyl-Lewis x glycoconjugate, to which theSulfo-SBED crosslinker (Pierce, Rockville, Ill.) had been conjugated,according to the manufacturers recommendations. The photo reactivecrosslinker group was activated by extensive UV irradiation (12-15hours), and then the biotin (re)tagged proteins were purified withstreptavidin coated magnetic beads as described before (Ilver-Arnqvist,et al., 1998). The extracted biotin tagged proteins were then separatedon SDS-PAGE, the 66 kDa band was digested with Trypsin (seq grade,Promega, U.S.A.) and analyzed on a Micromass TOF-Spec E (Micromass,Manchester, England), according to Larsson, et al., 2000. ProFound(www.proteometrics.com) was used for matching peptide masses (at NCBI).Peptide identities were validated by Q-TOF (Micromass), using thenanospray source, according to Nörregaard Jensen et al., 1999. Mascot(www.matrixscience.com) identified all our peptide sequences in thededuced amino acid sequence of JHP622 (FIG. 2 ;B) (SEQ ID NO: 5). SEQ IDNO: 1, QSIQNANNIELVNSSLNYLK, JHP622 aa 68-87 in FIG. 2;B, Grey bar. SEQID NO: 2, IPTINTNYYSFLGTK, JHP622 aa 625-639 in FIG. 2;B, Black bar. SEQID NO: 3, YYGFFDYNHGYIK, JHP622 aa 505-517 in FIG. 2;B, Black bar. SEQID NO: 4, DIYAFAQNQK, JHP622 aa 306-315 in FIG. 2;B, Grey bar.

[0036] Construction of the sabA-Mutant Strain

[0037] The J99 strain (Alm et al., 1999) was used for the constructionof the J99sabA(JHP662)::cam- and the J99/sabB(JHP659);cam-mutantstrains. The JHP662 gene was amplified using the F18 and R17 primers andcloned in pBluescript SK±EcoRV site, linearized with R20+F21 and ligatedwith the camR gene (Wang and Taylor, 1990). The JHP659 gene wasamplified using the F16+R15 primers and cloned in pCR2.1-TOPO vector(Invitrogen, Groningen, Holland), linearized with HincII and ligatedwith the camR gene. The H. pylori transformants were analyzed forbinding to ¹²⁵I-labeled sialyl-Lewis x glycoconjugate and the locationof the camR gene in JHP662 and JHP659 was analysed using the primersR17+F18 and F16+R15, respectively, where the mutants provided larger PCRproducts compared to the J99-stain. The sequences of the primers are asfollows: R15: CTATTCATGTTTACAATA; SEQ ID NO: 7 F16:GGGTTTGTTGTCGCACCACTAG; SEQ ID NO: 8 R17: GGTTCATTGTAAATATAT; SEQ ID NO:9 F18: CGATTCTATTAGATCACCC; SEQ ID NO: 10 R20: AGCGTTCAATAACCCTTACAGCG;SEQ ID NO: 11 F21: GATTTAAATACTGGCTTAATTGCTCG; SEQ ID NO: 12 BS22:CGCTTAAAGCATTGTTGACAGCC; SEQ ID NO: 13

[0038] Background Results and New Results

[0039] The Lewis b antigen binding adhesin, BabA, was recentlyidentified (Ilver-Arnqvist et al., 1998). We then analyzed thebabA-mutant strain, devoid of Lewis b antigen binding properties, forbinding to human gastric mucosa, and the babA-mutant strain demonstratesan adherence pattern most comparable to the CCUG17875 parent strain(denoted 17875). Thus, we then constructed the babA1A2-(double) mutantstrain, where both babA-genes were inactivated, since the tenaciousadherence observed by the babA2 mutant strain could possibly have beenascribed to recombination of the remaining silent babA1 gene intoexpression loci. However, the adherence pattern of the babA1A2-mutantstrain was still most similar to the 17975 (parent) strain. As expected,pre-treatment of the 17875 strain with soluble Lewis b antigen resultedin >80 % reduction of bacterial adherence to the epithelial cell lining.In contrast, adherence by the babA1A2-mutant strain was not affected.Screening of receptors for the babA1A2-mutant strain was performed bybinding of H. pylori and mAbs to panels of glycosphingolipids (GSLs)using the thin-layer chromatogram (HPTLC) binding technique (Ångström etal., 1998). The babA1A2-mutant strain differed from the parent 17875strain since the mutant does not recognize the Lewis b GSL. Instead, thebabA1A2-mutant strain recognizes acidic GSLs from human granulocytes andadenocarcinoma cells. Binding to these GSLs was abrogated by removal ofthe sialic acid residues. By probing the HPTLC-plates with thesialyl-Lewis x mAb, a staining pattern almost parallel to the bindingpattern of the babA1A2-mutant stain was obtained. High affinity GSLswere isolated from human adenocarcinoma tissue using the babA1A2-mutantstrain as a probe. The novel H. pylori receptor, thesialyl-dimeric-Lewis x GSL demonstrated high affinity for thebabA1A2-mutant strain (published in WO 00/56343).

[0040] Clinical isolates of H. pylori were analyzed by bindingexperiments to a series of soluble semi-synthetic glycoconjugates.Several combinations of adherence modes were found where the 17875strain binds the Lewis b antigen only, while the babA1A2-mutant strainbinds sialylated antigens. In our hands, the 26695 strain (genomesequenced by Tomb et al., 1997) binds neither antigen. In contrast, theJ99 strain (genome sequenced by Alm et al., 1999) recognizes both theLewis b and the sialyl-Lewis x (sLex) antigen (FIG. 1A, and published inWO 00/56343).

[0041] The prevalence of binding to the sialyl-Lewis x antigen wasassessed among Swedish clinical H. pylori isolates and 39% were foundpositive for binding. In comparison, 67% of the isolates bind the Lewisb antigen (Ilver-Arnqvist et al., 1998), and a majority of strains, 30out of the 39 isolates bind both the Lewis b and the sLex antigen.Interestingly, 15 out of the 39 sLex antigen binding strains also bindthe related sialyl-Lewis a antigen. (published in WO 00/56343, withsmall adjustments).

[0042] A Strong Correlation Found Between Sialidase DependentHemagglutination (HA) and Sialyl-Lewis x Antigen Binding

[0043] It has been known for more than a decade that H. pyloridemonstrates sialidase dependent hemagglutination (HA), i.e. aggregationdependent on sialylated glycoconjugates on the red blood cells (Evans etal., 1988). Thus, our panel of clinical strains were subjected to HA and27% (27/101) were found to provide positive HA-Titers. A strongcorrelation was found between HA titers and sialyl-Lewis x antigenbinding (FIG. 1B), which suggests that previous results on HA titers ofH. pylori strains, might actually relate to their ability for bindinginflammation associated sLex-antigens.

[0044] Human gastric mucosa have also been analyzed for expression ofsialylated glycoconjugates that promote adherence of H. pylori.Pretreatment of the babA1A2-mutant strain with the sLex conjugateabolished adherence (>90% reduction) to the gastric epithelial lining.In contrast, adherence by the 17875 parent strain was unaffected bysoluble sLex conjugate. The results strongly suggest that sLex antigenspromote adherence of H. pylori to the surface mucous cells in the humangastric epithelial lining (published in WO 00/56343). Non-H.pylori-infected, i.e., healthy Lewis b mouse gastric mucosa was analyzedfor expression of sialylated glycoconjugates, that promote adherence ofH. pylori. Pretreatment of the babA1A2-mutant strain with the sLexconjugate abolished adherence (>90% reduction) also to the Lewis b mousegastric epithelial lining. In contrast, adherence by the 17875 parentstrain was unaffected by soluble sLex conjugate. The results suggestthat sLex antigens confer adherence of H. pylori to the surface mucouscells in the Lewis b mouse gastric epithelial lining (published in WO00/56343)

[0045] Identification of the Corresponding Sialic Acid Binding Adhesin,SabA, a BabA-Related Member of the H. pylori Outer Membrane Protein(Hop) Family.

[0046] During the last decade various H. pylori proteins have beenproposed as sialic acid binding adhesins or hemagglutinins (reviewed inGerhard et al., 2001). Nevertheless, in an attempt to sort this out, wedecided to identify the corresponding sLex antigen binding adhesin.Since the adhesin activity was characterized by the promisingcombination of high binding specificity and high affinity for the sLexantigen, our recently developed Retagging technique would be the bestoption for the task. Retagging is based on the use of a multifunctionalbiotinylated crosslinker agent chemically attached to the receptor(Ilver-Arnqvist et al., 1998). Thus, for the present Retagg experimentswe used the sialyl-Lewis x conjugate. Since the affinity for the sLexantigen was lower compared to the previously described Lewis bantigen-BabA-interaction (Ilver-Arnqvist et al., 1998), the Retaggingprotocol was improved by use of extensive UV-exposure (see M&M). Theresulting Retagging (contact dependent biotin tagging of thecorresponding ligand protein) demonstrated a band of approx. 66 kDa onSDS gel (FIG. 2;A), which was analyzed by Maldi TOF. Four peptides wereidentified and mapped by computer analyzes to deduced amino acidsequences of the gene JHP662 in the J99 strain, but two out of the fourpeptides also matched the closely related deduced amino acid sequence ofJHP659 (Astra/Alm et al 1999) (FIG. 2;B), i.e. theQSIQNANNIELVNSSLNYLK-peptide (grey bar in FIG. 2;B)(SEQ ID NO: 1) andthe DIYAFAQNQK-peptide (grey bar in FIG. 2;B)(SEQ ID NO:4) are uniquefor the SabA protein (expressed by the JHP622 gene). The JHP662 andJHP659 genes are postulated outer membrane proteins with no knownfunction. A gene knockout of JHP662 completely abrogated all bindingactivity for the sLex antigen. In contrast, binding activity wasunperturbed by inactivation of JHP659 in the J99 strain. Thus JHP662,which corresponds to HP0725 in the 26695 strain (TIGR/Tomb et al.,1997), constitutes the gene that encodes the sialic acid bindingadhesin, SabA of the present invention, while the protein encoded by theJHP659/HP0722 genes was denoted SabB.

[0047] Summary of Results

[0048] The fucosylated blood group antigens, H1 and Lewis b, mediatebacterial adherence t the stomach epithelial and mucus lining (Borén etal., 1993). We recently identified the corresponding blood group antigenbinding adhesin, BabA (Ilver-Arnqvist, et al., 1998), by the Retaggingtechnique, based on the use of multfunctionell crosslinker structures.The clinical significance of the BabA adhesin is interesting, since itis highly associated with a virulent subset of H. pylori strains, the“triple-positive” strains (Gerhard et al., 1999). The present series ofexperimental results are based on the use of our defined babA mutantstrain, which does not bind the Lewis b antigen, but demonstrates analternative adherence mode for targeting the gastric epithelial lining.

[0049] A high affinity glycosphingolipid (GSL) was recently identifiedas the sialyl-dimeric-Lewis x antigen. The prevalence of bindingactivity among Swedish clinical isolates was then assessed, and 39% ofstrains bind the sialyl-Lewis x (sLex) antigen, compared to 67% ofstrains that bind the Lewis b antigen. H. pylori has actually for longbeen known to demonstrate sialic acid dependent adhesive properties(Evans et al., 1988). Here, among the Swedish strains, 27% demonstratesuch sialidase dependent hemagglutination (HA), and a strong correlationto sLex binding was found (FIG. 1;B ), which suggests that thecorresponding adhesins are interchangeable or identical.

[0050] The sialyl-Lewis x and sialyl-Lewis a antigens have previouslyboth been defined as inflammation markers and tumor antigens (Sakamotoet al., 1989; Takada et al., 1993). The binding of 15% of H. pyloristrains also to the sialyl-Lewis a antigen is intriguing considering thesialyl-Lewis a antigen both a tumor antigen (Magnani et al., 1981), andgastric dysplasia marker (Sipponen et al., 1986; Farinati et al., 1988),especially in relation to H. pylori as a possible carcinogen (IARCWorking Group on the Evaluation of Carcinogenic Risks to Humans, 1994).Recently, high level expression of the sialyl-dimeric-Lewis x antigenwas found to correlate with poor outcome in gastric cancer (Amado etal., 1998). Blood group O phenotype and non-secretor status areindependent risk factors for peptic ulcer disease (Sipponen et al.,1989). Non-secretor individuals lack the ABO blood group antigens (andthe Lewis b antigen) in secretions, such as saliva, and, in addition, inthe gastro-intestinal lining, where instead the Lewis a antigen and thesialyl-Lewis a antigens dominate (Sakamoto et al., 1989). In thisrespect it could be speculated that differences in adherence modes amongH. pylori strains could promote differences in disease outcome, as areflection of both individual blood group phenotype and secretor status.

[0051] The bacterial adherence properties were recently analyzed inrelation to the mucosal inflammation response of the correspondingtissue and significant correlation was found between sLex antigendependent adherence of the babA-mutant strain and (1) elevated levels ofinflammatory cell infiltration (2) sialyl-Lewis x antigen expression,and (3) histological gastritis (published in WO 00/56343).

[0052] Recently, increased expression of the sialyl-Lewis a antigen wasalso demonstrated in H. pylori infected individual and the sialyl-Lewisa antigen was expressed in fewer epithelial cells after H. pylorieradication (Ota et al., 1998). Similarly, the sialyl-Lewis x antigenwas found to be over-expressed in bronchial mucins from Pseudomonasaeruginosa-infected patients with chronic bronchitis (Davril et al.,1999). Thus, up-regulation of sialyl-Lewis antigens as a dynamicresponse to infectious agents could be a process similar to theestablished inflammation triggered expression of binding sites forselectin molecules in the endothelial cell lining (reviewed by Varki,1994). In the inflamed gastric mucosa, the stimulated up-regulation ofsialyl-Lewis antigen expression would then be available to H. pylori forsequential adherence modes. Thus, initial targeting to the epitheliallining by the virulent triple-positive strains would be directed by theLewis b antigen (Gerhard et al., 1999), while the sialyl-Lewis xglycosphingolipids would mediated subsequent establishment of intimatecontact with the cell membrane. Taken together, these results help outto understand the previous observations that chronic atrophic gastritisand dysplasia promote expression of sialylated structures (Sipponen etal., 1986), and that H. pylori demonstrate sialic acid dependenthemagglutination properties (Evans et al., 1988).

[0053] Here, the corresponding SabA adhesin, SEQ was purified bysialyl-Lewis x antigen primed Retagging technique, and the correspondingsabA gene was identified. The sabA gene is similar to the babA/B genesmembers of the Hop-family, i.e. the H. pylori outer membrane proteinwhich all demonstrate extensive homologies in the NH₂-terminal andCOOH-terminal domains (Tomb et al., 1997), where SabA and BabAdemonstrate 60% similarities in the N-terminal domain, 77% similaritiesin the 300aa C-terminal domain, but only 32% similarities in the centralregion (19% identities). However, the Hop proteins were recentlyphylogenetically mapped on the basis of the homologous C-terminaldomains, by Alm, et al., 2000. In this phyl-tree, the sabA adhesin gene(HP0725/JHP662/Hop P) and the closely related HP0722/JHP659/Hop O (inanalogy denoted sabB)), map next to the Lewis b antigen binding BabA/Badhesin genes (Hop S and T, respectively), and in addition, next to therecently postulated HopZ adhesin (Alm, et al., 2000). It is tempting tospeculate that the additional genes clustered in this distinct branchingof the Hop-phylogeny tree constitute the adhesin repertoire of H. pylorifor interaction with blood group antigen derived carbohydrates. Geneticrecombination and frame shifting events would allow the easy switchingon or off of adherence properties (Haas et al., 1986). Recombinationwithin the sabA and sabB genes could also provide the potential topromote flexible presentations of adhesive modes such as adaptation tofine tuned differences in the presentation of sialylatedglycoconjugates, such as affinity for the sialyl-Lewis x-versus thesialyl-Lewis a-antigens.

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1 13 1 20 PRT Helicobacter pylori 1 Gln Ser Ile Gln Asn Ala Asn Asn IleGlu Leu Val Asn Ser Ser Leu 1 5 10 15 Asn Tyr Leu Lys 20 2 15 PRTHelicobacter pylori 2 Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe LeuGly Thr Lys 1 5 10 15 3 13 PRT Helicobacter pylori 3 Tyr Tyr Gly Phe PheAsp Tyr Asn His Gly Tyr Ile Lys 1 5 10 4 10 PRT Helicobacter pylori 4Asp Ile Tyr Ala Phe Ala Gln Asn Gln Lys 1 5 10 5 651 PRT Helicobacterpylori 5 et Lys Lys Thr Ile Leu Leu Ser Leu Ser Leu Ser Leu Ala Ser Ser1 5 10 15 eu Leu His Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln20 25 30 Ile Gly Glu Ala Val Gln Met Val Lys Asn Thr Gly Glu Leu Lys Asn35 40 45 Leu Asn Glu Lys Tyr Glu Gln Leu Ser Gln Tyr Leu Asn Gln Val Ala50 55 60 Ser Leu Lys Gln Ser Ile Gln Asn Ala Asn Asn Ile Glu Leu Val Asn65 70 75 80 Ser Ser Leu Asn Tyr Leu Lys Ser Phe Thr Asn Asn Asn Tyr AsnSer 85 90 95 Thr Thr Gln Ser Pro Ile Phe Asn Ala Val Gln Ala Val Ile ThrSer 100 105 110 Val Leu Gly Phe Trp Ser Leu Tyr Ala Gly Asn Tyr Leu ThrPhe Phe 115 120 125 Val Val Asn Lys Asp Thr Gln Lys Pro Ala Ser Val GlnGly Asn Pro 130 135 140 Pro Phe Ser Thr Ile Val Gln Asn Cys Ser Gly IleGlu Asn Cys Ala 145 150 155 160 Met Asn Gln Thr Thr Tyr Asp Lys Met LysLys Leu Ala Glu Asp Leu 165 170 175 Gln Ala Ala Gln Gln Asn Ala Thr ThrLys Ala Asn Asn Leu Cys Ala 180 185 190 Leu Ser Gly Cys Ala Thr Thr GlnGly Gln Asn Pro Ser Ser Thr Val 195 200 205 Ser Asn Ala Leu Asn Leu AlaGln Gln Leu Met Asp Leu Ile Ala Asn 210 215 220 Thr Lys Thr Ala Met MetTrp Lys Asn Ile Val Ile Ala Gly Val Ser 225 230 235 240 Asn Val Ser GlyAla Ile Asp Ser Thr Gly Tyr Pro Thr Gln Tyr Ala 245 250 255 Val Phe AsnAsn Ile Lys Ala Met Ile Pro Ile Leu Gln Gln Ala Val 260 265 270 Thr LeuSer Gln Ser Asn His Thr Leu Ser Ala Ser Leu Gln Ala Gln 275 280 285 AlaThr Gly Ser Gln Thr Asn Pro Lys Phe Ala Lys Asp Ile Tyr Ala 290 295 300Phe Ala Gln Asn Gln Lys Gln Val Ile Ser Tyr Ala Gln Asp Ile Phe 305 310315 320 Asn Leu Phe Ser Ser Ile Pro Lys Asp Gln Tyr Arg Tyr Leu Glu Lys325 330 335 Ala Tyr Leu Lys Ile Pro Asn Ala Gly Lys Thr Pro Thr Asn ProTyr 340 345 350 Arg Gln Glu Val Asn Leu Asn Gln Glu Ile Gln Thr Ile GlnAsn Asn 355 360 365 Val Ser Tyr Tyr Gly Asn Arg Val Asp Ala Ala Leu SerVal Ala Lys 370 375 380 Asp Val Tyr Asn Leu Lys Ser Asn Gln Thr Glu IleVal Thr Thr Tyr 385 390 395 400 Asn Asn Ala Lys Asn Leu Ser Gln Glu IleSer Lys Leu Pro Tyr Asn 405 410 415 Gln Val Asn Thr Lys Asp Ile Ile ThrLeu Pro Tyr Asp Gln Asn Ala 420 425 430 Pro Ala Ala Gly Gln Tyr Asn TyrGln Ile Asn Pro Glu Gln Gln Ser 435 440 445 Asn Leu Ser Gln Ala Leu AlaAla Met Ser Asn Asn Pro Phe Lys Lys 450 455 460 Val Gly Met Ile Ser SerGln Asn Asn Asn Gly Ala Leu Asn Gly Leu 465 470 475 480 Gly Val Gln ValGly Tyr Lys Gln Phe Phe Gly Glu Ser Lys Arg Trp 485 490 495 Gly Leu ArgTyr Tyr Gly Phe Phe Asp Tyr Asn His Gly Tyr Ile Lys 500 505 510 Ser SerPhe Phe Asn Ser Ser Ser Asp Ile Trp Thr Tyr Gly Gly Gly 515 520 525 SerAsp Leu Leu Val Asn Phe Ile Asn Asp Ser Ile Thr Arg Lys Asn 530 535 540Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Ile Gln Leu Ala Gly Thr 545 550555 560 Thr Trp Leu Asn Ser Gln Tyr Met Asn Leu Thr Ala Phe Asn Asn Pro565 570 575 Tyr Ser Ala Lys Val Asn Ala Ser Asn Phe Gln Phe Leu Phe AsnLeu 580 585 590 Gly Leu Arg Thr Asn Leu Ala Thr Ala Lys Lys Lys Asp SerGlu Arg 595 600 605 Ser Ala Gln His Gly Val Glu Leu Gly Ile Lys Ile ProThr Ile Asn 610 615 620 Thr Asn Tyr Tyr Ser Phe Leu Gly Thr Lys Leu GluTyr Arg Arg Leu 625 630 635 640 Tyr Ser Val Tyr Leu Asn Tyr Val Phe AlaTyr 645 650 6 638 PRT Helicobacter pylori 6 Met Lys Lys Thr Ile Leu LeuSer Leu Ser Leu Ser Leu Ala Ser Ser 1 5 10 15 Leu Leu His Ala Glu AspAsn Gly Phe Phe Val Ser Ala Gly Tyr Gln 20 25 30 Ile Gly Glu Ala Val GlnMet Val Lys Asn Thr Gly Glu Leu Lys Asn 35 40 45 Leu Asn Asp Lys Tyr GluGln Leu Ser Gln Ser Leu Ala Gln Leu Ala 50 55 60 Ser Leu Lys Lys Ser IleGln Thr Ala Asn Asn Ile Gln Ala Val Asn 65 70 75 80 Asn Ala Leu Ser AspLeu Lys Ser Phe Ala Ser Asn Asn His Thr Asn 85 90 95 Lys Glu Thr Ser ProIle Tyr Asn Thr Ala Gln Ala Val Ile Thr Ser 100 105 110 Val Leu Ala PheTrp Ser Leu Tyr Ala Gly Asn Ala Leu Ser Phe His 115 120 125 Val Thr GlyLeu Asn Asp Gly Ser Asn Ser Pro Leu Gly Arg Ile His 130 135 140 Arg AspGly Asn Cys Thr Gly Leu Gln Gln Cys Phe Met Ser Lys Glu 145 150 155 160Thr Tyr Asp Lys Met Lys Thr Leu Ala Glu Asn Leu Gln Lys Ala Gln 165 170175 Gly Asn Leu Cys Ala Leu Ser Glu Cys Ser Ser Asn Gln Ser Asn Gly 180185 190 Gly Lys Thr Ser Met Thr Thr Ala Leu Gln Thr Ala Gln Gln Leu Met195 200 205 Asp Leu Ile Glu Gln Thr Lys Val Ser Met Val Trp Lys Asn IleVal 210 215 220 Ile Ala Gly Val Thr Asn Lys Pro Asn Gly Ala Gly Ala IleThr Ser 225 230 235 240 Thr Gly His Val Thr Asp Tyr Ala Val Phe Asn AsnIle Lys Ala Met 245 250 255 Leu Pro Ile Leu Gln Gln Ala Leu Thr Leu SerGln Ser Asn His Thr 260 265 270 Leu Ser Thr Gln Leu Gln Ala Arg Ala MetGly Ser Gln Thr Asn Arg 275 280 285 Glu Phe Ala Lys Asp Ile Tyr Ala LeuAla Gln Asn Gln Lys Gln Ile 290 295 300 Leu Ser Asn Ala Ser Ser Ile PheAsn Leu Phe Asn Ser Ile Pro Lys 305 310 315 320 Asp Gln Leu Lys Tyr LeuGlu Asn Ala Tyr Leu Lys Val Pro His Leu 325 330 335 Gly Lys Thr Pro ThrAsn Pro Tyr Arg Gln Asn Val Asn Leu Asn Lys 340 345 350 Glu Ile Asn AlaVal Gln Asp Asn Val Ala Asn Tyr Gly Asn Arg Leu 355 360 365 Asp Ser AlaLeu Ser Val Ala Lys Asp Val Tyr Asn Leu Lys Ser Asn 370 375 380 Gln ThrGlu Ile Val Thr Thr Tyr Asn Asp Ala Lys Asn Leu Ser Glu 385 390 395 400Glu Ile Ser Lys Leu Pro Tyr Asn Gln Val Asn Val Thr Asn Ile Val 405 410415 Met Ser Pro Lys Asp Ser Thr Ala Gly Gln Tyr Gln Ile Asn Pro Glu 420425 430 Gln Gln Ser Asn Leu Asn Gln Ala Leu Ala Ala Met Ser Asn Asn Pro435 440 445 Phe Lys Lys Val Gly Met Ile Ser Ser Gln Asn Asn Asn Gly AlaLeu 450 455 460 Asn Gly Leu Gly Val Gln Val Gly Tyr Lys Gln Phe Phe GlyGlu Ser 465 470 475 480 Lys Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe AspTyr Asn His Gly 485 490 495 Tyr Ile Lys Ser Ser Phe Phe Asn Ser Ser SerAsp Ile Trp Thr Tyr 500 505 510 Gly Gly Gly Ser Asp Leu Leu Val Asn PheIle Asn Asp Ser Ile Thr 515 520 525 Arg Lys Asn Asn Lys Leu Ser Val GlyLeu Phe Gly Gly Ile Gln Leu 530 535 540 Ala Gly Thr Thr Trp Leu Asn SerGln Tyr Met Asn Leu Thr Ala Phe 545 550 555 560 Asn Asn Pro Tyr Ser AlaLys Val Asn Ala Ser Asn Phe Gln Phe Leu 565 570 575 Phe Asn Leu Gly LeuArg Thr Asn Leu Ala Thr Ala Lys Lys Lys Asp 580 585 590 Ser Glu Arg SerAla Gln His Gly Val Glu Leu Gly Ile Lys Ile Pro 595 600 605 Thr Ile AsnThr Asn Tyr Tyr Ser Phe Leu Gly Thr Lys Leu Glu Tyr 610 615 620 Arg ArgLeu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 625 630 635 7 18 DNAHelicobacter pylori 7 ctattcatgt ttacaata 18 8 22 DNA ArtificialSequence Description of Artificial Sequence primer 8 gggtttgttgtcgcaccact ag 22 9 18 DNA Artificial Sequence Description of ArtificialSequence primer 9 ggttcattgt aaatatat 18 10 19 DNA Artificial SequenceDescription of Artificial Sequence primer 10 cgattctatt agatcaccc 19 1123 DNA Artificial Sequence Description of Artificial Sequence primer 11agcgttcaat aacccttaca gcg 23 12 26 DNA Artificial Sequence Descriptionof Artificial Sequence primer 12 gatttaaata ctggcttaat tgctcg 26 13 23DNA Artificial Sequence Description of Artificial Sequence primer 13cgcttaaagc attgttgaca gcc 23

1. Isolated Helicobacter pylori protein binding to sialyl-Lewis xantigen and having an approximate molecular weight of 66 kDa andcomprising the amino acid sequences SEQ ID NO: 1, QSIQNANNIELVNSSLNYLK,SEQ ID NO: 2, IPTINTNYYSFLGTK, SEQ ID NO: 3, YYGFFDYNHGYIK, and SEQ IDNO: 4, DIYAFAQNQK,

and sialyl-Lewis x antigen-binding H. pylori alleles of the protein,recombinant forms of the protein or the protein alleles, andsialyl-Lewis x antigen-binding portions of the proteins.
 2. Proteinaccording to claim 1, wherein the recombinant protein has the amino acidsequence SEQ ID NO:
 5. 3. Protein or a sialyl-Lewis x antigen bindingportion of the protein according to claim 1 or 2 for use as amedicament.
 4. Diagnostic antigen for the immunological determination,in a biological sample, of antibodies against sialyl-Lewis xantigen-binding protein, wherein the diagnostic antigen is an optionallylabeled protein or a sialyl-Lewis x antigen binding portion of a proteinaccording to claim 1 or
 2. 5. A method of determining the presence ofsialyl-Lewis x antigen-binding H. pylori bacteria in a biologicalsample, which comprises an immunological determination of the presenceof antibodies binding to an optionally labeled protein according toclaim 1 or
 2. 6. DNA molecule encoding a protein or a sialyl-Lewis xantigen binding portion of a protein according to claim 1 or
 2. 7.Vector comprising a DNA molecule according to claim
 6. 8. Hosttransformed with a vector according to claim
 7. 9. Method of determiningthe presence of sialyl-Lewis x or related carbohydrate structures in asample, comprising bringing the sample into contact with an optionallylabelled protein or sialyl-Lewis x antigen binding portion of a proteinaccording to claim 1 or 2, allowing binding of the protein orsialyl-Lewis x antigen binding portion of the protein according to claim1 or 2 to the carbohydrate structure and determining the presence ofsialyl-Lewis x or related carbohydrate structures in the sample bydetermining a) the occurrence of the binding, or b) the absence ofbinding in case an analyte inhibiting the binding is present.